This research will use genetic engineering techniques to examine the importance of compartmentation in control of metabolism in eucaryotic cells. Communication among central metabolic pathways in the cytosol (glycolysis and gluconeogenesis), in mitochondria (tricarboxylic acid cycle) and in peroxisomes (glyoxylate cycle in yeast) is accomplished through transport of a select number of metabolites across cellular membranes. Our goal is to determine the role of isozymes of malate dehydrogenase in regulating metabolic flux within and across compartmental barriers. This proposal focuses on essential features required for efficient function in different metabolic pathways. In initial experiments, the growth phenotypes and metabolic defects associated with loss of each isozyme will be determined following construction of Saccharomyces cerevisiae strains containing all possible combinations of disruptions in genes encoding mitochondrial (MDH1), cytosolic (MDH2), and peroxisomal (MDH3) malate dehydrogenases. Possible alternative compartmental sources of oxaloacetate will be tested with MDH mutants by disruption of genes encoding enzymes with related metabolic functions. Subsequent experiments will test the extent that structural divergence reflects efficiency for function in a specific metabolic pathway. To achieve this, the compartmental locations of the malate dehydrogenase isozymes will be altered by addition or removal of structural information for organellar targeting. Efficient function in an alternative pathway can then be tested by complementation of yeast mutants lacking the authentic compartmental isozymes. Other experiments will employ random mutagenesis and differential screening strategies to identify specific structural elements of the mitochondrial enzyme required for function in the tricarboxylic acid cycle and in the malate/aspartate shuttle cycle. Finally, structural elements of the cytosolic and peroxisomal enzymes which contribute to the rapid glucose- induced degradation of these enzymes will be determined and mechanisms for degradation in different organelles compared.